extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine Chemistry

text:
Down-regulation of osteopontin attenuates breast tumour progression in vivo

Development of breast tumour malignancies results in enhanced expression of various oncogenic molecules. Elevated expression of osteopontin (OPN) in higher grades of breast carcinoma correlates with enhanced expressions of several oncogenic molecules (urokinase-type plasminogen activator [uPA], matrix metalloproteinase-2/-9 [MMP-2 and -9]) and increased angiogenic potential of breast carcinoma. In this study, using in vitro and multiple in vivo models, we have demonstrated that silencing of OPN by its specific small interfering RNA (siRNA) down-regulates the expressions of oncogenic molecules such as uPA, MMP-2 and -9 resulting in inhibition of in vitro cell motility and in vivo tumourigenicity in mice. Moreover our results demonstrated that OPN−/− mice showed slower progression of tumour growth in breast cancer model as compared to wild-type mice. Furthermore, the data showed that injection of carcinogenic compound, pristane (2, 6,10,14-tetramethylpen-tadecane) induces breast tumour progression leading to enhanced expression of OPN and other oncogenic molecules in mammary fat pad of nude- and wild-type mice but not in OPN−/ mice. However, intratumoural injection of OPN siRNA to pristane-induced tumour significantly suppressed these effects. Our data revealed that knocking down of OPN effectively curb breast cancer progression and further suggested that developing of OPN-based therapeutics might be an emerging approach for the next generation of breast cancer management.

plays crucial role in activation of various kinases followed by NF-B and activator protein (AP)-1 activation that ultimately triggers downstream effector gene expression, which controls breast cancer progression [2,3]. In this study, we report that enhanced level of OPN expression in higher grades of breast tumours correlates with increased NF-B activation, enhanced serine and tyrosine phosphorylation, which further correlated with elevated activation of matrix metalloproteinase-2/-9 (MMP-2/-9) and expression of urokinase-type plasminogen activator (uPA).
Several studies have indicated that small interfering RNA (siRNA) targeting to vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and other oncogenic molecules results in significant suppression of tumour progression [14][15][16][17][18]. Filleur et al. have reported that intratumoural injection of VEGF siRNA significantly slowed tumour vascularization and growth, which further indicated the potential use of in vivo siRNAbased approach in cancer therapeutics [14]. Accordingly, we hypothesized that intratumoural injection of siRNA specific to OPN could attenuate breast tumour growth in nude mice model.
Recent reports indicated that OPN produced either from tumour or stroma has been shown to enhance the metastatic potential of transformed cells [19]. The decreased rate of tumour invasion to bone was shown in OPN knockout mice as compared to wild-type mice [20]. Moreover recent finding indicated that OPN-knockout mice exhibit a significant delay of tumour development [21]. Therefore we sought to determine whether there is reduced breast tumour growth and neovascularization in OPN -/mice compared to wild-type mice.
Here, using multiple in vitro and in vivo approaches, we have showed that OPN plays significant role in mammary carcinogenesis and our experimental data significantly correlated with the analysis of human breast clinical studies. Moreover, our study revealed that in vivo injection of OPN siRNA drastically reduced tumour growth. Furthermore, we have shown that the mice lacking OPN exhibit significant reduction of tumour growth. These data demonstrated, at least in part, that OPN could be a potential prognostic marker for the breast cancer progression and acts as key regulator of several other oncogenic molecules and further suggested that targeting OPN might be a novel approach for the next generation of treatment of breast cancer.

Materials
The rabbit anti-OPN polyclonal antibody was purchased from R & D Systems (Minneapolis, MN, USA). The anti-OPN antibody was also raised against purified intact human OPN in rabbit and characterized in our laboratory as described earlier [30]. Mouse

Human breast cancer specimen analysis
Human female breast tumour specimens and normal breast tissues were obtained from a local hospital with informed consent. Tissues were fixed in formaldehyde and paraffin blocks were prepared using standard protocols. Paraffin sections were used for histopathology and immunohistochemistry studies. Tumour grading was carried out by a modified Scarff-Bloom-Richardson (SBR) system with the help of an expert oncopathologist. Ten breast carcinoma specimens from each grade were used for analysis. Fresh tissues of various grades were also collected and used for electrophoretic mobility shift assay (EMSA).

OPN-specific siRNA duplex
The siRNA duplexes targeting human OPN and scrambled siRNA are described in Table 1

Western blot analysis and EMSA
Western blot and EMSA were performed as described [33,34].

Wound migration assay
Wound assay was performed using transfected or non-transfected MDA-MB-231 cells as described [7].

Gelatin zymography
To determine the gelatinolytic activity of MMP-2 and MMP-9, gelatin zymography was performed using tumour tissue lysates as described earlier [35].

In vivo xenograft model
MDA-MB-231 (5 ϫ 10 6 ) cells were injected orthotopically into the left inguinal mammary fat pad of female nude mice (NMRI) (n = 6/group). After 1 week, OPN-specific siRNA (250 g/kg body weight/mouse) was mixed with siRNA transfection reagent and injected intratumourally as described [14,36], thrice in a week until the completion of experiments. Control group of mice was injected with transfection reagent along with control OPNi 1 (Coni). Animals were kept in pathogen free conditions and monitored regularly. After 8 weeks, mice were sacrificed by cervical dislocation and photographed. Primary breast tumours were dissected out and weighed. One part of the tumours were fixed in 10% formalin solution and used for histopathology and immunohistochemistry. The other part was snap frozen and used for EMSA.

Generation of breast tumour in wild-type and OPN knockout mice
Mouse breast adenocarcinoma C127I (5 ϫ 10 6 ) cells were mixed with 500l cold growth factor depleted Matrigel and injected orthotopically to the left inguinal mammary fat pad of female wild-type (OPN +/+ ) and knockout (OPN -/-) mice (n = 6/group). The mice were kept in the pathogen-free condition for 8 weeks. After termination of experiments, mice were sacrificed; photographed, primary breast tumours were dissected out, weighed and analysed as described earlier.

Generation of pristane-induced mammary tumourigenesis in mice
Pristane was injected in the left inguinal mammary fat pad of female nude mice, thrice in a week for up to 6 weeks. After that, mouse OPN siRNA (mOPNi) (250 g/kg of body weight/mouse) mixed with transfection reagent was injected directly into the pristane-induced tumour thrice in a week for subsequent 6 weeks as described above. After 12 weeks, mice were sacrificed and mammary fat pad was dissected out and used for histopathology and immunohistochemical analysis.
In separate experiments, pristane was injected (thrice in a week) to the left inguinal mammary fat pad of female wild-type (OPN +/+ ) and knockout (OPN -/-) mice (n = 6/group). Mice were kept until development of tumour (16 weeks) and then sacrificed, photographed and tumours in mammary fat pad were dissected out for further analysis.

Immunofluorescence analysis
Immunofluorescence studies of human and mice tumours were performed as described earlier [34,37].

Statistical analysis
The bands were analysed densitometrically (Kodak Digital Science, USA) and fold changes were calculated. The expressions of OPN, uPA, MMP-2, MMP-9 and vWF in human clinical specimens were quantified using the Image Pro Plus 6.0 software (Nikon, Japan) and expressed as mean Ϯ SE. The mouse tumour weights were measured and plotted in the form of a bar graph. Statistical differences were determined by Student's t-test. Differences were considered significant when the P-value was less than 0.05.

Expression profiles of OPN, NF-B, uPA, MMP-2 and -9, MT1-MMP and vWF in different grades of human breast cancer specimens and its correlation with tumour progression and angiogenesis
The various histologic grades of breast carcinomas provide clinically important prognostic information [38]. The expression profile of different genes is related to the molecular basis of histological grades and it helps to improve the prognosis of breast cancer [38]. Because OPN is considered as a metastasis-associated gene and plays a crucial role in determining the oncogenic potential of several cancers including breast [2,3], we sought to determine the expression profiles of OPN and other oncogenic molecules in different grades of human breast carcinoma. Breast tumour clinical specimens were collected from a local hospital with informed consent and their gradations were analysed by a modified SBR system (n = 10/group). Figure 1A,  We have examined the cellular localization of NF-B in different grades of breast tumour tissues. In normal breast tissue or lower grades, distinct cytoplasmic localization of p65 subunit of NF-B was observed (Fig. 1A, panel V, as indicated by red arrows) whereas in grade III specimens, significant nuclear localization of p65 was found (as shown by white arrows). In grade II specimens, both cytoplasmic and nuclear localization of p65 was observed. The NF-B-DNA binding was determined by EMSA, which correlates with immunofluorescence study (Figs. 1A and C). These data suggested the correlation between enhanced OPN expression and NF-B activation, which is associated with enhanced malignancies in breast carcinoma. It has been reported that OPN induces AP-1 activation in breast cancer cells [2]. In this study, we observed the elevated AP-1-DNA-binding in higher grades of specimens (Fig. 1D). The expression profiles of uPA, MT1-MMP, MMP-2 and MMP-9 were analysed by immunohistochemistry by using their specific antibodies (Fig. 1A, panels VI-IX) and represented in the form of a bar graph (Fig. 1B). The elevated expressions of these molecules were observed in higher grades of tumours compared to normal tissue or lower grade of tumours. Interestingly, we have observed that the expression profiles of uPA and MMP-9 were considerably higher in grades II and III, whereas the enhanced levels of MMP-2 and MT1-MMP were detected in grade III tumours. The enhanced expression of vWF in grades II and III tumours suggested the increased angiogenic potential in higher grades of tumours (Fig. 1A, panel X and Fig. 1B)

Silencing of tumour-derived OPN suppresses the activation and expression of NF-B, AP-1, ERK, Akt, MMP-2, MMP-9 and uPA and inhibits breast cancer cell motility in vitro
To examine the role of tumour-derived OPN in regulation of downstream signalling molecules and cell motility, MDA-MB-231 cells were transfected with three different OPN siRNAs (OPNi) designed from three specific regions as described in Table 1. The OPN expression was determined by Western blot and the data indicated that OPNi 1 but not OPNi 2 and OPNi 3 specifically suppressed OPN expression ( Fig. 2A). To further examine the specificity of OPNi 1, single and double mismatched OPN siRNAs containing either one or two central mismatch was synthesized (Table 1). Our data revealed that single and double mutants were unable to suppress OPN expression, which further showed the specificity of OPNi 1 (Fig. 2B)

Silencing tumour-derived OPN abrogates breast tumour growth and MMP-2, MMP-9 and uPA expression in nude mice model
To investigate the role of tumour-derived OPN in the in vivo xenograft tumour growth, MDA-MB-231 cells were implanted in the mammary fat pad of female nude mice. OPNi and Coni (i.e. Coni1) were injected intratumourally as described in 'Materials and methods'. After 8 weeks, mice were sacrificed and the tumours were dissected out, photographed, weighed and analysed statistically and represented in the form of a bar graph (*P < 0.02) (Fig. 3A). The data indicated that OPNi significantly reduced the breast tumour load as well as growth characteristics in nude mice. Figure 3B, panels I and II, showed the typical histopathological morphology of mouse mammary tumours (10ϫ and 60ϫ magnification). The data indicated that xenograft tumours exhibit poorly differentiated structure in mammary fat pad. However, well-differentiated lobular structures were observed in OPNi-injected mammary fat pad of xenograft tumours (shown by blue arrows) indicating that silencing tumour-derived OPN reduces tumourigenicity. The characteristics of mice xenograft tumours are summarized in Table  2. Immunohistochemical analysis indicated that the expression profiles of OPN, MMP-2, MMP-9, MT1-MMP and uPA were significantly reduced in OPNi-injected xenograft tumours (Fig. 3B, panels III and IV and Fig. 3C, panels I-III). Interestingly, the phosphory-lation (serine and tyrosine) status of xenograft tumours of mice was drastically down-regulated in presence of OPN siRNA (Fig. 3C, panels IV and V). We have demonstrated earlier in this paper that increased serine and tyrosine phosphorylation correlates with enhanced tumourigenicity in human breast carcinoma specimens (Fig. 1A, panels III and IV). These data suggested that suppressing tumour-derived OPN not only down-regulates expressions of uPA and MMP-2/-9 but also inhibits total phosphorylations of tyrosine and serine residues of signalling molecules, which ultimately curb breast tumour progression. Moreover, our data also showed that there was reduction of nuclear localization of NF-B in the OPNiinjected tumours (Fig. 3C, panel VI). EMSA data support these findings and showed that OPNi reduced NF-B and AP-1-DNA binding (Fig. 3D, panels I and II). Recent studies have revealed that OPN plays crucial role in tumour angiogenesis [3]. Therefore to determine whether silencing of OPN results in suppression of angiogenesis in breast tumour model, the mice xenograft tissue sections were stained with anti-vWF (an endothelial cell specific marker) antibody and visualized under confocal microscopy. The results indicated that vWF expression in xenograft tumour was drastically reduced in presence of OPNi (Fig. 3C, panel VII). These results clearly demonstrated that tumour-derived OPN plays crucial role in regulation of tumour progression and angiogenesis because targeting OPN by its specific siRNA suppresses the expression of oncogenic molecules and down-regulates the neovascularization that ultimately abrogates tumour progression.  to the left inguinal mammary fat pad of wild-type and OPN -/mice. Mice were kept for 8 weeks and then sacrificed. The data indicated that there were significant reductions of breast tumour growth in OPN -/mice (>5.5 fold) as compared to wild-type mice (Fig. 4A). These data suggested that host OPN also played important role in regulation of breast tumour growth. Histopathological analysis of tumour tissue sections showed that there were significantly higher cellular infiltration, nuclear polymorphism and poorly

Fig. 3 OPN-specific siRNA suppresses breast tumour progression and angiogenesis in nude mice. (A) MDA-MB-231 (5 ϫ 10 6 ) cells were injected orthotopically into the left inguinal mammary fat pad of female nude mice (NMRI) (n = 6/group). After 1 week, OPN-specific siRNA (250 g/kg body weight/mouse) was mixed with siRNA transfection reagent and injected intratumourally, thrice in a week until the completion of experiments. Control group of mice was injected with transfection reagent along with control OPN siRNA 1 (Coni). Typical photographs of orthotropic xenograft tumours are shown (upper panel). Isolated tumours are shown in inset. Note that intratumoural injection of OPNi but not Coni significantly reduced tumour load. Lower panel: The tumour weights were quantified and represented in the form of a bar graph (*P < 0.02) (n = 6). (B and C) Histopathology (haematoxylin and eosin) of the mice breast tumour section. Photographs were taken in 10ϫ and 60ϫ magnification (B, panels I and II). Blood vessels in the tumour section were indicated by black arrows whereas the typical breast lobular structure in OPNi-injected tumours were indicated by blue arrow. Expression profiles of OPN, MMP-2, MMP-9, MT1-MMP and uPA were detected by immunohistochemical studies (B, panels III and IV and C, panels I-III). OPN, MMP-9 and uPA were stained with Cy-3 whereas MT1-MMP was stained with FITC. Nuclei were stained with DAPI. Status of total phosphorylations were shown using anti-phosphoserine and anti-phosphotyrosine antibodies followed by staining with FITC-conjugated IgG (C, panels IV and V). Cellular localization of NF-B, p65 and expression of vWF (neovascularization) were shown by immunofluorescence using their specific antibodies followed by staining with Cy3 conjugated IgG (panels VI and VII). (D) EMSA analysis of NF-B (panel I) and AP-1 (panel II)-DNA binding in tumour tissues.
differentiated structure in tumour of wild-type mice as compared to OPN -/mice (Fig. 4B, panels I and II). The expressions of MMP-9, MMP-2, uPA and MT1-MMP were also detected by immunohistochemistry and the data showed that the expression levels of all these molecules are significantly down-regulated in OPN -/mice (Fig. 4B, panels IV-VII). Moreover, reduction in tumour vascularization was observed in OPN -/mice (Fig. 4B, panel VIII). The level of OPN expression in tumour lysates was detected by Western blot and the gelatinolytic activity of MMP-2 and MMP-9 were examined by zymography. The data showed that there were significant reductions of OPN expression (Fig. 4C) (Fig. 4D). These data indicated that lack of host OPN expression resulted in reduced expressions of various oncogenic molecules such as MMPs, uPA and down-regulated the activations of NF-B and AP-1 that ultimately suppressed breast tumour growth and angiogenesis.

Exposure with external carcinogen is known to induce tumours.
Recently it has been reported that carcinogenic compound such as asbestos induces expression of OPN in patients with pleural mesothelioma [39]. To examine the role of external carcinogen in mammary tumourigenesis, we injected pristane, a known natural carcinogen to the mammary fat pad of nude mice and observed that there was significant induction of tumour growth. To determine the role of OPN in pristane-induced breast tumourigenesis, mOPNi was injected intratumourally into the pristane-induced tumours and the data showed that OPNi inhibits pristane-induced mammary tumourigenesis. Tumour weights were measured and the data indicated that injection of mOPNi reduced more than 60% of tumour weight in mice (data not shown). The mammary tumours were also analysed by histopathology. The micrographs of the tumour sections were taken under microscope (10ϫ and 60ϫ magnifications) (Fig. 5A, panels I and II). The characteristics of these tumours are described in Table 3. The injection of pristane into the mammary fat pad of nude mice showed poorly differentiated structure with high nuclear polymorphism and large number of mitotic features (Table 3). Enhanced vascularization was observed in pristane induced but not in OPNi-injected tumours (Fig. 5A, (Fig. 5A, panel III). These data indicated that injection of pristane resulted in enhanced breast tumour load in nude mice whereas direct delivery of OPNi to the site of pristane-induced tumours showed reduction of the breast tumour growth (Table 3).

panels II, indicated by arrows). Immunohistochemical studies with anti-OPN antibody showed that there was marked induction of OPN expression in pristane-induced tumours
These findings prompted us to analyse the expression and activation status of various oncogenic molecules and transcriptions factors in these tumour samples. The results indicated the high levels of MMP-9, MMP-2, MT1-MMP and uPA expressions in pristane-induced tumours compared to normal mammary fat pad of nude mice (Fig. 5A, panels IV-VI and Fig. 5B, panel I).
OPNi drastically suppressed the pristane-induced expression of these molecules in tumours. Furthermore, the data suggested that pristane not only induced the nuclear localization of NF-B, p65 (indicated by arrows) but also enhanced its DNA binding (Fig. 5B, panel II and Fig. 5C, upper panel). OPNi suppressed the pristane-induced NF-B nuclear translocation, NF-B and AP-1-DNA binding (Fig. 5B, panel II and Fig. 5C, upper and lower panels). Moreover, the total phosphorylations of serine and tyrosine residues of signalling molecules were significantly higher in pristane-induced tumours compared to control or OPNi-injected tumours (Fig. 5B, [2]. Therefore, we sought to determine the status of phosphorylations of these kinases in pristane-induced mammary tumours and our data revealed that there were marked inductions of phosphorylations of ERK and Akt in pristane-induced tumours but not in OPNi-injected tumours (Fig. 5B, panels V and VI). Elevated expression of vWF was also observed in pristine-induced mammary tumours (Fig. 5B, panel VII). However, OPNi inhibits vWF expression in these tumours, which further indicated that pristane promotes OPN-dependent mammary tumourigenesis through the induction of angiogenesis.

Reduced progression of pristane-induced tumour growth and angiogenesis in OPN-deficient (OPN -/-) mice
Our previous findings prompted us to determine the effect of pristane on breast tumourigenesis in wild type (wt) and OPN -/-mice. Accordingly, pristane was injected in the mammary fat pad of wt and OPN -/mice. After termination of experiments, mice were sacrificed. Figure 6A shows the typical photographs of mice with breast tumours. Our data revealed that there was significant reduction of breast tumour growth (~65%) in OPN -/mice as compared to wt mice (Fig. 6A). The tumour sections were analysed histopathologically and the data indicated that there was significant loss of tumourigenicity (less infiltration, well-differentiated structure, reduced mitotic feature) in the OPN -/mice, whereas enhanced tumourigenesis was observed in pristane-induced wt mice (i.e. enhanced infiltration, poorly differentiated structure, more mitotic feature, enhanced vasculature) (Fig. 6B, panels I and II). The level of OPN expression was almost absent in tumours of OPN -/mice (Fig. 6B, panel III). The pristane-induced tumours in wild-type but not OPN -/mice were positive to CA-15-3 (breast tumour marker) (Fig. 6B, panel IV) suggesting that these are indeed breast tumours. The expressions of MMP-9, uPA and vWF were significantly higher in tumours of wt mice as compared to OPN -/mice (Fig. 6B, panels V-VII). The gelatinolytic activity of MMP-2 and MMP-9 were detected by zymography from the tumour lysate (Fig. 6C). The data showed that there was significant reduction of MMP-2 and MMP-9 activation in pristane-induced tumours of OPN -/mice as compared to wild-type mice (Fig. 6C). Enhanced NF-B and AP-1-DNA bindings were also observed in wild-type but not in OPN -/mice (Fig. 6D). Taken together, our data indicated that pristaneinduced breast tumours were significantly suppressed in absence of host OPN, which indicated that OPN plays crucial roles in this process.

Discussion
Tumour progression is a series of complex events that requires the interplay among various cytokines, kinases, transcription factors, proteases and oncogenic molecules. Recent studies have indicated that OPN acts as a key regulator of tumour progression through activating several signalling cascades, growth factors and inducing the expression of several oncogenic molecules [2,3]. Thus we hypothesized that targeting OPN might be a novel approach to suppress the metastatic potential of cancers. In this study, we showed that silencing of OPN expression significantly down-regulates breast tumour progression in xenograft models. Moreover, we observed the higher expression of OPN in pristaneinduced tumours and silencing of OPN expression by OPNi significantly abrogates pristane-induced mammary tumourigenesis in nude mice models. Interestingly, the breast tumour model in OPN -/mice showed slower progression of tumour growth as compared to wt mice. Using four independent mice models, we have demonstrated that OPN either produced in tumour and/or stromal environment plays a crucial role in regulation of breast tumour growth and angiogenesis.
Although various therapeutic approaches have been recently implicated for the anti-cancer therapy, there are several accidental side effects of these approaches (i.e. chemotherapy, radiotherapy, etc.) that may cause severe physiological disorder which might reduce the chances of survival of the patients. Therefore, the recent concept of cancer therapy is directed towards targeting the molecule that is involved in regulation of metastatic signature or angiogenic switch [40][41][42]. Recent studies have suggested that expression profiling of various gene may provide the molecular basis of histological grade to improve prognosis and treatment of breast cancers [38]. Earlier reports have indicated that OPN acts as most potent metastasis-associated gene and plays important roles in regulation of tumour angiogenesis and considered as the potent oncogenic marker during breast cancer progression [3]. Thus targeting OPN at transcriptional or protein level or blocking its receptor or its downstream signalling pathways could be an emerging approach for anti-cancer therapy [43,44]. In recent times, intratumoural delivery of siRNA showed great prospect in cancer therapy particularly in in vivo mice model [14,36]. In this study, we design a small interfering RNA (siRNA) against OPN and demonstrate that intratumoural injection of siRNA significantly suppressed breast tumour growth and angiogenesis in the mouse model.
Exposure of the external carcinogen is one of the major causes of cancer development. It is well established that exposure of external carcinogens results in alteration of several gene expression and activation of oncogenes that ultimately induce cancer progression [45]. These reports prompted us to determine the effect of OPN-specific siRNA in carcinogen-induced tumourigenesis in nude mice. We found that prolonged and continuous pristane injection in the mammary fat pad resulted in development of breast tumour growth in nude mice. We have observed the enhanced expression of OPN and other oncogenic molecules such as MMPs and uPA and enhanced angiogenesis in pristane-induced tumours. Our data showed that direct delivery of OPN-specific siRNA significantly suppressed pristane-induced tumourigenesis. Moreover, we have for the first time demonstrated the reduction of pristine-induced tumour growth and angiogenesis in OPN knockout mice as compared to wild-type ones.
The role of host OPN in regulation of tumour growth in mice is not well defined. Previous studies have indicated that OPN deficiency in mice significantly reduced tumour metastasis in bone and lung [46]. Ohyama et al. has shown that deficiency of OPN in mice reduced the bone loss caused by implantation of melanoma cells into the bone marrow space [20]. In contrast, the data also showed that significant alteration of tumour metastasis in bone was not observed in OPN knockout mice [47]. Hsieh  in OPN -/mice [21]. The variation of these studies might be due to the nature of tumours or origin of OPN knock out mice. In this study, we have observed that there was reduced progression of breast tumour growth and angiogenesis in OPN -/mice.
Finally, in this study, we have demonstrated that targeting OPN might be specific and important therapeutic approach for the treatment of breast cancer. Moreover our findings suggested that pristane, a potent natural carcinogen induces breast cancer progression that could be characterized by enhanced OPN expression and silencing of this gene significantly suppresses cancer progression. Taken together, our study suggested that OPN is one of the major molecules that control the breast cancer progression. Thus, targeting OPN might be a novel approach for the next generation of breast cancer management.


extfieldsofstudy: Biology Medicine

text:
Requirement of histone deacetylase1 (HDAC1) in signal transducer and activator of transcription 3 (STAT3) nucleocytoplasmic distribution

Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that plays a crucial role in interleukin-6 (IL-6) signaling, mediating the acute-phase induction of the human Angiotensinogen (hAGT) gene in hepatocytes. We showed earlier that IL-6 induces acetylation of the STAT3 NH2-terminus by the recruitment of the p300 coactivator. We had also observed a physical interaction of STAT3 and Histone Deacetylase1 (HDAC1) in an IL-6-dependent manner that leads to transcriptional repression. In this study, we sought to elucidate the mechanism by which HDAC1 controls STAT3 transcriptional activity. Here, we mapped the interacting domains of both STAT3 and HDAC1 and found that the COOH-terminal domain of HDAC1 is necessary for IL-6-induced STAT3 transcriptional repression, whereas the NH2-terminal acetylation domain of STAT3 is required for HDAC1 binding. Interestingly, over expression of HDAC1 in HepG2 cells leads to significantly reduced amounts of nuclear STAT3 after IL-6 induction, whereas silencing of HDAC1 resulted in accumulation of total and acetylated STAT3 in the nucleus. We have found that HDAC1 knockdown also interferes with the responsiveness of the STAT3-dependent MCP1 target gene expression to IL-6, as confirmed by real-time RT–PCR analysis. Together, our study reveals the novel functional consequences of IL-6-induced STAT3-HDAC1 interaction on nucleocytoplasmic distribution of STAT3.

INTRODUCTION
The signal transducers and activators of transcription (STATs) are a family of latent cytoplasmic transcription factors mediating target gene activation in response to cytokines and growth factor stimulation (1,2). Seven STAT family members (and their alternative splice products) have been identified, with each member being activated by a distinct spectrum of cytokines (3). Like other STAT transcription factors, STAT3 is predominantly cytoplasmic in resting cells, a feature which facilitates the ability of STAT3 to directly transduce signals from cell surface associated cytokine receptor to target genes in the nucleus. In response to cytokine stimulation, STATs become tyrosine phosphorylated at their COOH-terminus by receptor and receptor-associated tyrosine kinases. Activated STATs form homo-or heterodimers through intermolecular src homology domain 2 (SH2)-phosphotyrosine interactions, disengage from the liganded receptor and subsequently translocate into the nucleus where they bind enhancer sequences [5 0 -TT(N4-6)AA-3 0 ] of target genes (4,5). Once the activated STAT dimer recognizes a target promoter, the transcription rate from this promoter is dramatically increased, reflecting the ability of STAT transcriptional activation domains to recruit nuclear coactivators that mediate chromatin decondensation and communicate with proteins binding the core promoter.
Although phosphorylation is a crucial posttranslational modification that regulates the activities of different proteins, there are many others including methylation (6), ubiquitination (7), sumoylation (8), isgylation (9) and acetylation (10). Indeed, it has been found that different STAT 1 and 3 isoforms are inducibly acetylated, a modification yielding a variety of consequences for target gene transcription. For example, we had recently shown that IL-6-induced acetylation of STAT3 NH 2 -terminus is required for recruitment of the p300 coactivator and is necessary for target gene expression through a novel mechanism involving acetylation/deacetylation (11,12). We further showed that the STAT3 NH 2 -terminal acetylation is necessary for target gene transcription by stabilizing the STAT3-p300 complex. Others have found that STAT3 dimerization is regulated by reversible acetylation of lysine *To whom correspondence should be addressed. Tel: +1 409 772 2824; Fax: +1 409 772 8709; Email: suray@utmb.edu ß 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
at 685 in the SH2 domain of STAT3 (13). STAT1 is also acetylated by the CBP coactivator, a modification that regulates NF-kB activity leading to induction of apoptosis (14). Together, these observations indicate that site-specific acetylation of STAT3 is an important regulatory modification that influences protein-protein interaction.
Histone and non-histone protein acetylation is a reversible reaction controlled by the steady state level of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In humans, HDACs are divided into three categories: class I RPD3-like proteins (HDAC1, HDAC2, HDAC3 and HDAC8); the class II HDA1-like proteins (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9 and HDAC10); and the class III SIR2-like proteins (15,16). Class I HDACs are ubiquitously expressed while the expression of many class II HDACs are tissue-specific. Recently class IV HDACs, a group consisting of HDA-C11-related enzymes has also been described (17,18). HDAC1, 2 and 8 are predominantly nuclear proteins while HDAC 3, 4, 5, 7 and 9 shuttle between the nucleus and cytoplasm (15).
The class I enzyme HDAC1 is a nuclear protein and can heterodimerize with the closely related deacetylase HDAC2 (19). Two important functional regions of HDAC1 proteins have been identified in mouse (20): the NH 2 -terminus contains a motif required for HDAC1 homo-oligomerization and for hetero-oligomerization of HDAC1 with HDAC2 and HDAC3. The COOH-terminal lysine rich sequence (amino acid residues 438-482), on the other hand, is crucial for the nuclear localization of HDAC1. Both HDAC1 and 2 are found in three major multiprotein complexes, named Sin3, NuRD, and CoREST (21,22). HDAC1 can repress gene transcription either directly or as part of these multiprotein complexes when recruited to the promoter by a variety of transcriptional regulators (15,23). One emerging concept is that acetylation-deacetylation reactions play a role in cellular processes independent of transcription. For example, acetylation regulates protein stability, protein-protein interactions and nuclear localization (24). Acetylation of HNF4 and E1A adenovirus-transforming protein, is known to enhance nuclear retention by increasing nuclear import and blocking nuclear export of these transcription factors (25,26). Conversely, deacetylation of SRY, a Y chromosome-encoded DNA-binding protein, by HDAC3 induces cytoplasmic delocalization of the protein from the nuclear compartment (27). HDAC mediated deacetylation of proteins, in many cases is known to be prerequisite for subsequent ubiquitination and degradation (16,24).
In this study we show for the first time that acetylation of STAT3 and its subsequent binding with HDAC1 is involved in control of its nucleocytoplasmic distribution. We present evidence that the NH 2 -terminal domain of STAT3 interacts with HDAC1, and that STAT3 acetylation is required for this interaction. Overexpression of HDAC1 in HepG2 cells leads to significantly reduced amount of IL-6-induced nuclear STAT3, whereas HDAC1 silencing through RNA interference results in STAT3 accumulation in the nucleus. Consistent with this finding, we have found that HDAC1 knockdown also interferes with the responsiveness of the STAT3-dependent reporter gene and target gene expression to IL-6 stimulation. Together, these findings suggest a novel mechanism of how protein-protein interaction coupled with chemical modification regulate STAT3 transcription.

siRNA transfection
Control and HDAC1 siRNA (Dharmacon Smart Pools) were transfected into HepG2 cells by TransIT-siQUEST transfection reagent (Mirus, Madison, WI) at 50 nmol/l final concentration, following the manufacturer's instruction. After 48-72 h, cells were stimulated with IL-6 for different time periods as indicated.

Antibodies and immunoprecipitation
Sources of primary antibody (Ab) included Santa Cruz Biotechnology for anti-STAT3 (K15 and C20) and antiphospho-Tyr STAT3 (B7); New England Bio Labs for anti-HDAC1 Ab; anti-V5 and anti-FLAG Ab were obtained from Invitrogen and Sigma respectively. Antiacetyl K87STAT3 Ab was generated and affinity purified as described earlier (11).
For immunoprecipitation and Western blot analysis, either whole cell extracts (WCEs) or Nuclear Extracts (NEs) were used as indicated. WCEs were prepared by lysing HepG2 cells in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mM PMSF, 1 mM NaF, 1 mM Na 3 VO 4 , and 1 mg/ml each of aprotinin, leupeptin, and pepstatin). Sucrose cushion-purified NEs were prepared by lysing HepG2 cells with nonionic detergent (0.5% IGEPAL-60) and centrifugation over a sucrose cushion as described (28). Extracts were precleared with protein A-Sepharose 4B (Sigma) for 10 min at 48C and the cleared lysate incubated with primary Ab for 2-12 h at 48C. Immune complexes were captured by adding 30 ml of protein A-Sepharose beads (50% slurry) and rotated for 1 h at 48C. Beads were washed three times for 5 min with cold PBS, immune complexes eluted by incubation in SDS-PAGE loading buffer and fractionated by 10% SDS-PAGE. Proteins were transferred to polyvinylidine difluoride membranes (Millipore, Bedford, Mass.) and blocked with 5% milk for 1 h followed by primary antibody treatment for overnight at 48C. Membranes were washed in TBST (0.1%) tween and incubated with secondary antibody for 1 h. Signals were detected by the enhanced chemiluminescence assay (ECL, Amersham) or visualized by the Odyssey Infrared Imaging system (LICOR Biosciences, Lincoln, NE).

Real-time RT-PCR analysis
Total cellular RNA was extracted by Tri Reagent (Sigma). One microgram of RNA was used for reverse transcription using iScript cDNA Synthesis Kit (Bio Rad). Three microliters of cDNA products was amplified in 20 ml reaction system containing 10 ml iQ SYBR Green Super Mix (Bio Rad) and 400 nM primer mix. All the primers were designed by Primer Express version 2.0 software. For Human MCP-1 mRNA expression, forward primer 5 0 -CATTGTGGCCAAGGAGATCTG-3 0 and reverse primer 5 0 -CTTCGGAGTTTGGGTTTGCTT-3 0 were used. All reactions were processed in MyiQ Single-Color Real-Time PCR Detection System (Bio Rad) and results were analyzed by IQ5 program (Bio Rad). To normalize template input, GAPDH (endogenous control) transcript level was measured for each sample. Data are expressed as fold change relative to unstimulated, after normalizing to GAPDH.

Immunofluorescence staining
Cells were acetone-methanol fixed, rinsed in PBS containing 50 mM NH 4 Cl for 10 min, and permeabilized in PBS containing 0.1% Triton X-100. Cells were then blocked with pre-immune heterologous serum (diluted 1 : 10 in PBS) for 30 min, washed, and incubated with primary and secondary (FITC and Rhodamine (Rd)-conjugated) Ab. After staining, cells were mounted in antifading mounting solution (DAKO Inc. Carpentaria, CA). Confocal microscopy was performed on a Zeiss LSM510 META System using the 488 nm line of the Argon-laser for excitation of FITC and Helium-Neon 543 nm line for excitation of Rd, combined with appropriate dichroic mirrors and emission band filters to discriminate between green and red fluorescence. Images were captured at a magnification of Â60 (60Â oil immersion objective numerical aperture 1.4). Co-localization was visualized by superimposition of green and red images using MetaMorph software Version 4.6r9 (Universal Imaging Corp).

In vitro deacetylation assay
Wild type (aa 1-770) and NH 2 -terminal deleted (aa 130-770) V5-tagged STAT3 plasmids were expressed in HepG2 cells and in vitro deacetylation assay (UPSTATE Biochemicals) was performed following the protocol as recommended by the supplier.
To gain further insights into how IL-6-induced STAT3-HDAC1 interaction could control target gene repression, we examined the effect of increasing doses of ectopically expressed HDAC1 on IL-6 inducible transcription using a high affinity STAT3-binding site termed (hAPRE1) 5 -LUC reporter plasmid, in HepG2 cells (28). Relative to empty vector control, cells transfected with HDAC1 showed significant inhibition of IL-6-mediated hAGT promoter activity in a dose-dependent manner ( Figure 1A). A significant reduction of promoter activity was seen with transfection of only 0.2 mg of HDAC1 expression plasmid and a >90% reduction with 0.8 mg. This confirms our previous observation of the inhibitory effect of HDACs in IL-6-induced transactivation (11).

The COOH-terminal domain of HDAC1 is required for target gene repression
To identify the domains required for transcriptional inhibition, FLAG epitope-tagged HDAC1 deletion mutants were expressed and effects were tested on STAT3-dependent transcription. For this purpose, HepG2 cells were cotransfected with (hAPRE1) 5 -LUC reporter plasmid and expression plasmids encoding either with FLAGtagged HDAC1 full length (FL, aa 1-482) or the HDAC1 NH 2 -terminal regulatory domain (ÁC, aa 1-140) or the HDAC1 carboxy-terminal domain (ÁN, aa 141-482). In absence of HDAC1, IL-6 strongly induced (hAPRE1) 5 -LUC reporter activity by 8-fold (Figure 2A). Cotransfection of HDAC1 full length strongly inhibited both basal and IL-6-induced reporter activity. By contrast, the HDAC1 NH 2 -terminal regulatory domain had little effect. Expression of the HDAC1 COOH-terminal decaetylase and phosphorylation domain showed a strong inhibitory effect on STAT3 dependent transcription. This result suggested to us that the histone deacetylation activity of HDAC1 is closely linked to its transcriptional repression activity.
We next performed coimmunoprecipitation studies to localize the domains of HDAC1 that bind to endogenous STAT3. FLAG-epitope tagged HDAC1 FL (aa 1-482), HDAC1 ÁC (aa 1-140) and HDAC1 ÁN (aa 141-482) were transfected in HCT 116 cells and IL-6 stimulated WCEs were immunoprecipitated with anti-FLAG conjugated agarose beads. Western immunoblot analysis with anti-STAT3 antibody shows that both NH 2 -terminal domain and COOH-terminal domain of HDAC1 bind to endogenous STAT3, although these domains had differential repression effect on STAT3 transcription ( Figure 2B).
NH 2 -terminal acetylation of STAT3 is necessary for HDAC1 binding Next, we tested the requirement of STAT3 NH 2 -terminal acetylation for its in vivo interaction with HDAC1 by transfecting HepG2 cells with a full-length STAT3 acetyldeficient mutant (STAT3 K49R/K87R). Figure 2C, lane 6  1 (HDAC1). HepG2 cells were transiently transfected with (hAPRE1) 5 -LUC reporter plasmid, pSV2-PAP internal control, and indicated amounts of HDAC1 expression vectors (200, 400, 600 and 800 ng, respectively). Twenty four hours after transfection, cells were stimulated with IL-6 (8 ng/ml). Luciferase activity was measured after 24 h of stimulation and normalized to alkaline phosphatase activity. Amount of transfected DNA was kept equivalent using an empty expression plasmid. Ã P < 0.05 relative to empty vector, t-test. Ectopic expression of FLAG-tagged HDAC1 in transient transfection is shown by Western blot (inset). (B) Transrepression of STAT3 with HDAC1. HepG2 cells were transfected with UAS-LUC and either Gal4-STAT3 (full length, 1-770) expression plasmid or Gal4-STAT3 and HDAC1 together and stimulated with IL-6. HDAC1 repressed transactivation of UAS-LUC by GAL4-STAT3. Luciferase activity was measured after 24 h of IL-6 stimulation and normalized to alkaline phosphatase activity. Ã P < 0.05 relative to empty vector, t-test. shows that efficient binding of HDAC1 with STAT3 requires acetylation of STAT3 since STAT3 K49R/K87R apparently weakly binds to endogenous HDAC1. However, because the expression of STAT3 K49R/K87R were reduced relative to those of wild type (WT) STAT3 we conducted further experiments with FLAG epitoped tagged NH 2 terminal domain of STAT3, where the level of expression was comparable. For this experiment, FLAG-WT STAT3 (1-124), pseudoacetylated STAT3 (K49/87Q) (1-124) and acetyl deficient STAT3 (K49/ 87R) (1-124) were transfected and complexes were immunoprecipitated with anti-FLAG agarose beads ( Figure 2D). We noted that endogenous HDAC1 bound with the WT-and pseudoacetylated STAT3, whereas the acetyl mutant STAT3 bound very weakly.
Next, we performed confocal immunohistochemistry to study colocalization of WT STAT3 and NH 2 -terminal deleted STAT3 (130-770) with endogenous HDAC1. Expression vectors encoding WT-STAT3 and STAT3 (130-770) were transfected in HepG2 cells and stimulated with IL-6. Both HDAC1 (red) and STAT3 (green) were distributed throughout the nucleus and are excluded from the nucleoli ( Figure 3A). The merged image showed that WT STAT3 colocalized with endogenous HDAC1, whereas the NH2-terminal deleted STAT3 mutant poorly localized, further confirming requirement of the NH2-terminal STAT3 in HDAC1 binding. Together, these results indicate that NH 2 -terminal domain of STAT3 as well as its acetylation is necessary for stable complex formation with HDAC1.
We next sought to determine whether the STAT3-HDAC1 complex is associated with HDAC activity. For this purpose, we developed an immunoprecipitationin vitro deacetylation assay. Wild type (aa 1-770) V5-tagged STAT3 and NH 2 -terminal deleted STAT3 (aa 130-770) were cotransfected with HDAC1 expression vector and IL-6 stimulated HepG2 WCEs were immunoprecipitated with V5 antibody. The washed immune complexes were then incubated in vitro with [ 3 H]-labeled histone H4 (aa 2-24) peptide and deacetylation activity was measured by [ 3 H] release. We observed strong histone deacetylase activity in the immunoprecipitated V5-tagged full length STAT3 complexes from IL-6 stimulated HepG2 cells. Most strikingly, the 130 aa NH 2terminal deletion of STAT3 (130-770), a mutation that does not bind HDAC1 ( Figures 2C, 2D and 3A), showed very little HDAC activity. This data further indicated that the STAT3 NH 2 -terminus is essential for the functional recruitment of histone deacetylases ( Figure 3B).

HDAC1 controls nuclear STAT3 abundance
To verify the role of HDAC1 in STAT3 transcriptional activity, IL-6 responses were evaluated in cells where HDAC1 expression level was either enhanced or reduced. For this, HepG2 cells were transfected with HDAC1 expression vector or empty vector as control and stimulated with IL-6 for 0, 20, 60 and 90 min. Nuclear and cytoplasmic distribution of endogenous STAT3 protein was then determined by Western immunoblot analysis with anti-STAT3 Ab (Figure 4). In empty vector transfected cells, IL-6 induced a rapid accumulation of STAT3 within 20 min of stimulation, concomitant with the nuclear appearance of phospho-Tyr 705 STAT3. In contrast, HDAC1 overexpression reduced nuclear STAT3 (total) abundance after IL-6 stimulation compared to empty vector control although the phospho-Tyr 705 STAT3 level remained the same. Because HDAC1 reduced the nuclear accumulation of STAT3 and enhanced its cytoplasmic abundance, these data suggested to us that HDAC1 is involved in STAT3 nucleocytoplasmic partitioning. with IL-6 for 30 min. Cells were fixed and incubated with anti-V5 Ab and antibody to HDAC1. Binding of primary antibody was detected by FITC-or Texas red-labeled secondary antibody. Confocal microscopy was performed on a Zeiss LSM510 META System using the 488 nm and 543 nm excitation for FITC and Texas red, respectively. Images were captured at a magnification of Â60. Co-localization was visualized (in the right panel, merge) by superimposition of green and red images using MetaMorph software. (B) Histone deacetylase activity is associated with STAT3 immune complex. HepG2 cells were transfected with either control empty vector, or full-length V5-tagged STAT3 or STAT3 NH 2 -terminal deleted mutant (aa 130-770) that weakly binds HDAC1. IL-6 stimulated WCEs were immunoprecipitated with anti-V5 Ab and these immune complexes were used for in vitro deacetylation assay to decaetylate [ 3 H]-labeled histone H4 peptide (substrate), as described in the deacetylation assay kit protocol (UPSTATE Biochemicals). Ã P < 0.05 relative to empty vector control extract, t-test.

HDAC1 knockdown accumulates acetylated STAT3 in the nucleus
To further prove the involvement of HDAC1 in STAT3 nucleocytoplasmic distribution, the effect of reducing endogenous HDAC1 level was investigated. For this purpose, RNA interference was used to reduce HDAC1 expression in HepG2 cells, where HDAC1 levels were reduced by more than 70%, compared to control siRNA ( Figure 5A). IL-6-dependent nuclear translocation of STAT3 was then determined by Western immunoblot analysis after HDAC1 knockdown. HepG2 cells were transfected with either control siRNA or HDAC1 specific siRNA and IL-6 stimulated prior to NE preparation. Western blots of NEs were probed with either anti-STAT3 Ab, or anti-phospho-STAT3 Ab or with antiacetyl K87 STAT3 Abs (11). Figure 5B indicates that, in the presence of control siRNA, STAT3 is efficiently translocated from cytoplasm into the nucleus. In presence of HDAC1 siRNA, both the total STAT3 and acetylated STAT3 accumulated in the nucleus even in the absence of stimulation. Interestingly, we did not observe any change in abundance of phospho-Tyr 705 STAT3 in the nucleus in the presence of HDAC1 siRNA. This above result of nuclear retention along with that from the HDAC1 overexpression study (Figure 4) strongly implicate that HDAC1 plays an important role in nucleocytoplasmic partitioning of STAT3.

Subcellular localization of STAT3 after HDAC1 knockdown
We next assessed the dynamics of cytoplasmic-nuclear translocation of STAT3 by confocal microscopic imaging on HepG2 cells transfected either with control siRNA or with HDAC1 siRNA, in presence or absence of IL-6. In control siRNA-treated unstimulated cells, the majority of STAT3 was detected in the cytoplasm ( Figure 6). Treatment of cells with IL-6 for 30 min altered the equilibrium between the nuclear and cytoplasmic STAT3 and induced a complete nuclear translocation of STAT3 and which again redistributed back into the cytoplasm after 90 min of stimulation. Similarly, with HDAC1 siRNA treatment, although we observed some basal level of STAT3 in the nucleus and in the perinuclear region of the resting cells, STAT3 rapidly translocated in the nucleus from cytoplasmic compartment after 30 min of IL-6 stimulation, and it remained accumulated in the nucleus even after 90 min of IL-6 treatment, a time when STAT3 normally exported back into the cytoplasm (upper panel). This data further supports our above conclusion that HDAC1 is involved in STAT3 nucleocytoplasmic partitioning.

HDAC1 knockdown interferes with the responsiveness of STAT3-dependent target gene activation
To determine HDAC1's role in IL-6 inducible transcription, cells transfected with HDAC1 siRNA were subsequently re-transfected with STAT3-dependent (hAPRE1) 5 -LUC reporter. As seen in Figure 7A, IL-6 treatment increased STAT3 dependent (hAPRE1) 5 -LUC luciferase activity by 10-12-fold in control siRNA  transfected cells. In the HDAC1 knockdown cells, however, IL-6 inducible reporter activity was further increased to 30-fold, indicating that endogenous HDAC1 levels normally repress IL-6-inducible transcription. We interpret these data to mean that the effect of HDAC1 knockdown on hAGT reporter is due to reduced nuclear export, leading to nuclear accumulation of acetyl STAT3 and enhanced target gene transcription.
Next, to determine whether the effect of HDAC1 on STAT3 transcription is cell type-specific or global phenomena, we measure more quantitatively the effect of HDAC1 knockdown on IL-6-inducible STAT3 target gene by real-time RT-PCR analysis. For this purpose we used human acute monocytic leukemia THP-1 cells (29) to measure the abundance of Monocyte Chemotactic Protein-1 (MCP1) mRNA after IL-6 induction for different time periods. MCP1 is known to be an IL-6-induced STAT3 downstream target gene and is expressed in human promonocytic cell lines (30). In contrast to control siRNA, the induction of MCP1 mRNA after HDAC1 knockdown was significantly increased after 24 h and 40 h of IL-6 treatment ( Figure 7B).
From these results, we concluded that HDAC1 is required for the termination of IL-6-dependent transcription by STAT3 and subsequent redistribution from the nucleus.

DISCUSSION
STAT3 is the central mediator of IL-6-induced gene expression which is phosphorylated at Tyr 705 by the receptor associated Janus kinases, an essential prerequisite for dimerization and nuclear translocation (1,3). In addition, an acetylation/deacetylation cascade of STAT3 has been recently identified (11,13), which is essential for its enhanced transcriptional competence. The duration and degree of gene activation in response to cytokine signals is primarily dictated by the subcellular distribution and nucleocytoplasmic shuttling of STAT3. STAT3 is predominately cyotplasmic in unstimulated resting cells. Upon cytokine stimulation, it quickly translocates to the nucleus, where it mediates the induction of immediate early genes. To control transient signal transmission by activated STAT3, re-export of STAT3 back to the cytoplasm is also regulated. Thus nuclear export of STAT3 serves to prepare the cells for its next round of signaling.  Forty-eight hours after transfection cells were stimulated with IL-6 for 24 h and then harvested for luciferase reporter assay. Ã P < 0.05 relative to control siRNA, t-test. (B) Silencing of HDAC1 activates IL-6 regulated MCP1 expression in human monocytic THP1 cells. THP1 cells were transfected with either control siRNA or HDAC1 siRNA. Thirty-six hours after transfection, cells were stimulated with IL-6 for indicated time and total RNA was extracted at the same time for real-time RT-PCR analysis. Ã P < 0.05 relative to control siRNA, t-test.
Although the mechanism controlling the nuclear import of STAT3 upon cytokine stimulation is known (2,3), the mechanism by which activated STAT3 export from the nucleus is not well defined. Previous studies have shown that nuclear STAT3 is regulated by two distinct export of pathways controlled by three distinct nuclear export signal (NES) elements with various potencies, located at residues 306-318 (NES1), 404-414 (NES2), and 524-535 (NES3) (31). Among these, NES1 and NES2 are conserved with STAT1, whereas NES3 being unique to the STAT3 isoform. NES1 appears to be important for rapid post stimulation nuclear export, whereas NES2 and NES3 play important roles in controlling the basal nuclear export pathways. Acetylation of histones and non-histone proteins are reversed by a large number of HDACs, which are ubiquitous in eukaryotic cells. Human HDAC1 and HDAC2 exist together in at least three distinct multi-protein co-repressors complexes called the Sin3, NURD/Mi2, and CoREST complexes (21,22). Many known transcriptional repressors such as YY1 and RB associate and recruit the HDAC1/HDAC2 and mSin3A corepressor complexes to target gene promoters, thereby repressing transcription (22). Although, majority of HDAC1 target genes showed reduced expression accompanied by recruitment of HDAC1 and local reduction in histone acetylation at regulatory regions, a small subset of HDAC1 target genes (Gja1, Irf1 and Gbp1) were also found that require HDAC1 activity and recruitment for their transcriptional activation (32). This regulation occurs either by direct recruitment of HDAC1 to the promoter or by indirect deacetylation of acetylated transcription factors thereby modulating their activity. Recently, posttranslationaly modified HDAC1 was found to associate with hormoneactivated glucocorticiod receptor (GR) (33). Here, hormone activation of GR leads to progressive acetylation of HDAC1, an event that inhibits the deacetylase activity of the enzyme required for promoter activation.
In contrast to our observation where HDAC1 acts as a repressor for IL-6-mediated STAT3 transactivation, it has also been demonstrated that the deacetylase activity of HDAC1 (and also 2 and 3) is required for STAT1 dependent gene activation (34,35). Treatment of HDAC inhibitors (HDACi), or silencing of HDAC1, 2 and 3 negatively regulate STAT1-dependent transcriptional activation. These findings suggest that hyperacetylation of histones may negatively regulate STAT1-dependent gene activation. Consistent with these findings, it has been recently demonstrated that STAT1 undergoes acetylation upon HDACi treatment in human melanoma cell lines, which alters its interaction with HDACs, CBP and NF-kB. Acetylated STAT1 can only interact with NF-kB p65 and inhibits its DNA binding, nuclear localization and hence expression of anti-apoptotic genes (14).
In this study, we provide the first evidence for the involvement of HDAC1 on nucleocytoplasmic distribution of STAT3. We have also delineated a molecular mechanism by which STAT3 acetylation-mediated HDAC1 interaction controls partitioning of STAT3. Our earlier studies showed that IL-6-dependent acetylation of two Lys residues (Lys 49 and Lys 87 ) in the STAT3 NH 2 -terminus plays a critical role in transactivation of the human AGT gene (11). We also showed that HDAC1 stably interacts with STAT3 in IL-6-dependent manner and downregulates hAGT transactivation. In this study, we found that the NH 2 -terminal domain (aa residues 1-130) of STAT3 is required for HDAC1 binding and acetylation of this domain at Lys 49 and Lys 87 are necessary for HDAC1 interaction. It is interesting to note that the NH 2 -terminus of STAT3 is an independently folded modular domain that is highly conserved across the various STAT family members (36). The NH 2 -terminus is thought to play an important role in DNA-binding and protein-protein interaction. For example, NH 2 -terminus of both STAT1 and STAT3 have been shown to interact with the p300 acetyltransferase (13,37). In our previous study we have found that NH 2terminal acetylation of STAT3 is also required for its stable interaction with p300 (11). This evidence indicates that the HDAC1 deacetylase and the p300 acetylase interact with STAT3 in the same NH 2 -terminal region (aa 1-130). Together, we suggest that HDAC1 negatively regulates STAT3 transcription not only by its catalytic activity indirectly, but also inhibiting interaction with p300 and playing a key role in STAT3 transcription.
We showed earlier that the acetylation deficient STAT3 mutant also has a qualitative defect in cytoplasmic redistribution (11). This raised the possibility that acetylation of STAT3 may controls its nucleocytoplasmic distribution. In this present study, our observation of acetylation-deficient mutant STAT3 had reduced binding with HDAC1 can explain our previous observation why the STAT3 acetyl mutant had delayed nuclear export.
Three different assays were used to investigate the role of HDAC1 in the nucleocytoplasmic partitioning of STAT3. First, over expression of HDAC1 in HepG2 cells, followed by IL6-stimulation, resulted in a rapid nuclear export and hence less nuclear STAT3 abundance. Whereas in empty vector transfection, STAT3 efficiently translocates into the nucleus within 20 min of IL-6 stimulation and starts to redistribute back into the nucleus in 60-90 min, the kinetics that are markedly accelerated with overexpressed HDAC1. Second, siRNA mediated knock down of endogenous HDAC1 prolonged STAT3 nuclear translocation. Here we found accumulation of total and AcSTAT3 in unstimulated NE (because of constitutive shuttling) and this level remained the same even after IL-6 stimulation. Interestingly, we did not observe any accumulation of phospho-Tyr 705 STAT3 in the nucleus of unstimulated cells indicating that dephosphorylated and acetylated STAT3 accumulated in the nucleus of resting cells in presence of HDAC1 siRNA. Although dephosphorylation of Tyr 705 of STAT3 was proposed to be involved in nuclear export to a certain extent (38,39), our observation that HDAC1 overexpression/knockdown does not change the phosphorylated form of STAT3 in the nucleus, indicates that HDAC1 is not involved in the dephosphorylation of STAT3. Third, immunofluoresence studies with HDAC1 knockdown cells showed a slightly increased basal amount of STAT3 in the nucleus of unstimulated cells which increased upon IL-6 stimulation (30 min). Importantly, there was a delayed re-export of nuclear STAT3 after 90 min of IL-6 stimulation. These data further support our conclusion that HDAC1 is required for STAT3 distribution.
STATs are nucleocytoplasmic shuttling proteins that uses of both carrier-independent and carrier-mediated translocation mechanism (40). The exportin CRM1 supports nuclear export of STATs to a minor extent (39,40). Functional studies with GFP-tagged STAT3 and previously identified NESs by Bhattacharya et al. indicated that these elements (NES1, 2 and 3) direct a bonafide CRM1-dependent nuclear export. The crystal structure of STAT3 bound to DNA indicated that the NES2 (404-414) and NES3 (524-535) is either buried or partially exposed. It is likely that these elements will only regulate export after the STAT complex is dissociated from DNA or binding of others factors that affect the conformation of the protein. Thus it appears likely that structural changes associated with binding of HDAC1 to AcSTAT3 may play an important role in binding of CRM1 with nuclear export signal (NES) (31). Alternatively, it is possible that after transactivation, AcSTAT3 binds with HDAC1 for deacetylation that enhances its interaction with nuclear exporter CRM1 or with other HDACs (HDAC3 or other type II HDACs that can shuttle between cytoplasm to nucleus (41,42). However, it is interesting to note that acetylation sites of STAT3 are not located in either of these NESes. Therefore, how the AcSTAT3-HDAC1 association couples with the rapid post-stimulation nuclear export pathway will require further investigation.
In conclusion, we provide evidence that HDAC1 regulates STAT3 nucleocytoplasmic distribution at least in part by binding with acetylated STAT3 in IL-6-dependent manner. Because STAT3 is of pathological importance, and HDAC inhibitors are emerging as promising therapeutic agent against numerous diseases, their ability to modify cytokine-dependent transcriptional regulation is an important clinical consideration that might guide the development of second generation deacetylase inhibitors.